The present invention relates primarily to improvements in methods of DNA sequencing. In particular, the invention relates primarily to the elimination of stops or pauses in chain termination methods of DNA sequencing by the addition of nitrogen-containing organic compounds such as betaine, trimethylamine N-oxide and dimethylglycine. The invention also provides for DNA sequencing kits containing these compounds. The invention also provides for improvements in other laboratory procedures using DNA polymerases, such as polymerase chain reaction (PCR).
Efficient DNA sequencing technology is very important to the development of the biotechnology industry as well as for basic biological research. Improvements in both efficiency and accuracy of DNA sequencing are needed to keep pace with the demands for DNA sequence information. The Human Genome Project, for example, has set a goal for dramatically increasing the efficiency, cost-effectiveness and throughput of DNA sequencing techniques. (See Collins, F., and Galas, D. (1993) Science 262:43-46.)
Most DNA sequencing today is carried out by a chain termination method of DNA sequencing. The most popular chain termination methods are variants of the dideoxynucleotide-mediated chain termination method of Sanger (see Sanger et al. (1977) Proc. Nat. Acad. Sci., USA 74:5463-5467). Thousands of laboratories employ this technique including those doing automated sequencing for the Human Genome Project. Commercial kits containing the reagents needed for this method of DNA sequencing are available and are widely used.
Although commonly used, the Sanger (dideoxy) sequencing technique has problems and limitations. One of the major problems with this method is the incidence of DNA polymerase stops or pauses which interfere with the determination of the DNA sequence. Stops are predominantly problematic in regions of the DNA that are GC-rich or in regions that are especially far from the primer. In addition, stops occur more frequently in impure DNA preparations. Because of this, DNA purification is generally required before DNA can be sequenced by the dideoxy method.
Various methods have been proposed to eliminate stops in dideoxy sequencing. For example, researchers have tried varying the reaction temperature, using a variety of DNA polymerases, stabilizing the DNA polymerase, and extending the prematurely terminated DNA molecules with terminal deoxynucleotidyl transferase (see T. W. Fawcette and S. G. Bartlett (1990) BioTechniques 9:46-48; D. Pisa-Williamson and C. W. Fuller (1992) United States Biochemical Corp. Comments 19:29-36; J. Sambrook, E. F. Fritsch and T. Maniatis, ed. (1989) Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; and F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl, ed. (1989) Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscientific, John Wiley and Sons, N.Y.) However, none of these methods has been reliable. There is a continuing need to eliminate the problem of stops in DNA sequencing and thereby improve the efficiency and cost-effectiveness of this important process.